The long-term goal of this work is to understand tRNA splicing in the yeast S. cerevisiae and vertebrates. tRNA splicing is essential in yeast, man and likely all eukaryotes, and is uniquely different from other classes of splicing. Superficially, tRNA splicing is simple in yeast: an endonuclease excises the intron, an RNA ligase joins the half-molecules to leave a splice junction 2'-phosphate, and a 2'-phosphotransferase transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate (Appr>p), a previously unknown metabolite. The enzymes are highly conserved in vertebrates, and the last step works in vivo in Xenopus oocytes. Yet there are striking layers of complexity: First, the formation of Appr>p in equimolar amounts during splicing implies a pathway to return it to known metabolism and suggests a regulatory function in yeast as a sensor. Second, vertebrates apparently also have a second ligase that has been implicated in tRNA splicing, which uses different chemistry and does not require a 2'-phosphotransferase. This is a rare occurrence of two seemingly redundant pathways in the same organism, and suggests some form of regulation, or another use of one of the pathways. Third, the ligase that catalyzes tRNA splicing is unexpectedly also required in yeast for HAC1 mRNA splicing to mediate the unfolded protein response, suggesting that this function of ligase is also possible in other eukaryotes. Fourth, a fully functional form of the 2'-phosphotransferase is found in E. coli, and evolutionary analysis indicates that it has been in bacteria for more than a billion years. This is a surprise, since E. coli is not known to have introns, and bacteria do not splice RNA by this mechanism. Its retention there suggests an important function unrelated to splicing which, by extension, may also occur in eukaryotes. Since the first step of the mechanism is strikingly similar to the ADP-ribosylation catalyzed by a number of toxins (such as Diphtheria and cholera), and overproduction of the bacterial protein causes a distinct growth defect, the enzyme may have important regulation function. This proposal aims to study: the role of Appr>p in the cell through modulation of its levels in yeast and analysis of cellular function; how tRNA splicing is catalyzed in vertebrates, by use of an assay that detects both pathways simultaneously, and by studying expression of mouse phosphotransferase in different tissues; and the role of the phosphotransferase in yeast and E. coli, by the construction and analysis of mutated phosphotransferase proteins, coupled with analysis of the growth defect and its likely cause in E. coli.